With the advent of recombinant DNA technology, the controlled microbial production of an enormous variety of useful polypeptides has become possible. Many mammalian polypeptides, such as leukocyte interferons, have already been produced by various microorganism strains. The power of the technology admits the microbial production of an enormous variety of useful polypeptides, putting within reach the microbially directed manufacture of hormones, enzymes, antibodies, and vaccines useful against a wide variety of diseases.
A basic element of recombinant DNA technology is the plasmid, a nonchromosomal loop of double-stranded DNA found in bacteria oftentimes in multiple copies per cell. Included in the information encoded in the plasmid DNA is that required to reproduce the plasmid in daughter cells (i.e., a "replicon") and ordinarily, one or more selection characteristics, such as resistance to antibiotics, which permit clones of the host cell containing the plasmid of interest to be recognized and preferentially grown in selective media. The utility of bacterial plasmids lies in the fact that they can be specifically cleaved by one or another restriction endonuclease or "restriction enzyme", each of which recognizes a different site on the plasmidic DNA. Thereafter heterologous genes or gene fragments may be inserted into the plasmid by endwise joining at the cleavage site or at reconstructed ends adjacent the cleavage site. (As used herein, the term "heterologous" refers to a gene not ordinarily found in, or a polypeptide sequence ordinarily not produced by, a given microorganism, whereas the term "homologous" refers to a gene or polypeptide which is found in, or produced by the corresponding wild-type microorganism.) DNA recombination is performed outside the microorganism, and the resulting "recombinant" replicable expression vehicle, or plasmid, can be introduced into microorganisms by a process known as transformation and large quantities of the heterologous gene-containing recombinant vehicle obtained by growing the transformant. Moreover, where the gene is properly inserted with reference to portions of the plasmid which govern the transcription and translation of the encoded DNA message, the resulting expression vehicle can be used to actually produce the polypeptide sequence for which the inserted gene codes, a process referred to as expression. Expression is initiated in a region known as the promoter. RNA polymerase recognizes a promter and binds to it prior to initiation of transcription. In some cases, as in the lac and trp systems discussed infra, promoter regions are overlapped by "operator" regions to form a combined promoter-operator. Operators are DNA sequences which are recognized by so-called repressor proteins which serve to regulate the frequency of transcription initiation in a particular promoter. In the transcription phase of expression, the RNA polymerase recognizes certain sequences in and binds to the promoter DNA. The binding interaction causes an unwinding of the DNA in this region, exposing the sense coding strand of the DNA as a template for initiated synthesis of messenger RNA from the 5' to 3' end of the entire DNA sequence. The messenger RNA is, in turn, bound by ribosomes within which the encoded message is translated into a polypeptide having the amino acid sequence for which the DNA codes. Each amino acid is encoded by a unique nucleotide triplet or "codon" which collectively make up the "structural gene", i.e., that part which encodes the amino acid sequence of the expressed polypeptide product.
After binding to the promoter, the RNA polymerase initiates first the transcription of nucleotides encoding a ribosome binding site including a translation initiation or "start" signal (ordinarily ATG, which in the resulting messenger RNA becomes AUG), then the nucleotide codons within the structural gene itself. So-called stop codons are transcribed at the end of the structural gene whereafter the polymerase may form an additional sequence of messenger RNA which, because of the presence of the stop signal, will remain untranslated by the ribosomes.
Ribosomes bind to the binding site provided on the messenger RNA, in bacteria ordinarily as the mRNA is being formed, and themselves direct the production of the encoded polypeptide, beginning at the translation start signal and ending at the previously mentioned stop signal(s). The desired polypeptide product is produced if all remaining codons follow the initiator codon in phase. The resulting product may be obtained by lysing the host cell and recovering the product by appropriate purification from other bacterial protein.
Polypeptides expressed through the use of recombinant DNA technology may be entirely heterologous, as in the case of the direct expression of human growth hormone, or alternatively may comprise a heterologous polypeptide and, fused thereto, at least a portion of the amino acid sequence of a homologous polypeptide, as in the case of the production of intermediates for somatostatin and the components of human insulin. In the latter cases, for example, the fused homologous polypeptide comprised a portion of the amino acid sequence for beta galactosidase. In those cases, the intended bioactive product is bioinactivated by the fused, homologous polypeptide until the latter is cleaved away in an extracellular environment. Fusion proteins like those just mentioned can be designed so as to permit highly specific cleavage of the precusor protein from the intended product, as by the action of cyanogen bromide on methionine, or alternatively by enzymatic cleavage. See, e.g., G.B. Patent Publication No. 2 007 676 A.
If recombinant DNA technology is to fully sustain its promise, systems must be devised which optimize expression of gene inserts, so that the intended polypeptide products can be made available in controlled environments and in high yields.
The lactose promoter/operator systems have been commonly employed but, while useful, do not fully utilize the capacity of the technology from the standpoint of yield. However, they have the distinct advantage of being very controllable, a characteristic which is very desirable where large-scale microbial fermentation production is concerned. The control mechanism lies in the mode of action of the operator. When the operator is in a repressed mode, because of bound repressor protein, the DNA dependent RNA polymerase is competively prevented from binding and initiating transcription. Thus, the transcriptional pathway is closed, and this effectively blocks promoter operability. This system can be derepressed by induction following the addition of a known inducer, such as isopropyl-beta-D-galactoside. (IPTG). The inducer causes the repressor protein to fall away so the RNA polymerase can function. Thus, these types of promoter/operators are said to be "inducible." Cells transformed with plasmids carrying the so-called lac promoter/operator system can be permitted to grow up to maximum density while maintaining the promoter/operator system in a repressed state simply by omission of an inducer, such as IPTG, in the fermentation culture. When a high level of cell density is achieved, the system can be derepressed by additon of inducer and the promoter is then free to initiate transcription so as to obtain optimal expression of the gene products at yields commensurate with the promoter strength. However, despite these advantages, certain of the inducible promoters, such as the lac promoter/operator system are relatively weak and productions utilizing such promoters do not urge the microorganism to generate maximum output.
As a response to the need for microbial expression vehicles capable of producing desired polypeptide products in higher yield, the so-called tryptophan promoter/operator was harnessed and is currently widely used. The tryptophan promoter/operator system is one of many known, relatively powerful systems--upwards of three to four times the strength of the lac promoter. Although the power of such systems is attractive for commercial production, the advantages of this power are somewhat offset by less promoter control. In many of these powerful systems, the operator is not inducible in the sense defined above. Instead, the bound repressor which closes down the promoter pathway is not removable by induction. Another system was devised whereby the attenuator region of the tryptophan promoter/operator system was removed and the cells transformed with this system grown up in the presence of tryptophan rich media. This provided sufficient tryptophan to essentially completely repress the operator so that cell growth could proceed uninhibited by premature expression of the desired heterologous polypeptide system. When the culture reached appropriate growth levels, no additional tryptophan was supplied, resulting in mild tryptophan limitation and, accordingly, derepression of the promoter with highly efficient expression of the heterologous insert. However ingenious, this system, on an industrial scale, does not have the refined control characteristics of a promoter system having an operator which can be completely repressed and derepressed by induction. In practice, for example, it is necessary to maintain high levels of tryptophan during the growing up phase to completely repress the promoter and to permit the medium to become exhausted of tryptophan following full growth of the culture. It is apparent, therefore, that a need has existed for a microbial promoter/operator system which is capable of highly controlled production of desired polypeptide products in high yield.
The present invention addresses the problems associated with present promoter/operator systems and uniquely provides new hybrid systems which exhibit substantial advantages. The present invention is further directed to a method of microbially producing heterologous polypeptides, directed by highly efficient and controlled promoter/operator systems, as well as associated means. It is particularly directed to the use of hybrid promoter/operator systems which are designed to function well in large, industrial scale production.